The present invention, in some embodiments thereof, relates to compositions and methods for diagnosing and treating B-CLL.
In normal individuals, the pool of peripheral lymphocytes is constant in size. The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, survival, and proliferation. Survival factors have been shown to play a critical role in maintaining lymphocyte homeostasis.
Chronic lymphocytic leukemia, the most common leukemia in the Western world, is characterized by the progressive accumulation of CD5+ small mature lymphocytes, in the peripheral blood, lymphoid organs and bone marrow. The hallmark of the disease is decreased apoptosis, resulting in accumulation of these malignant cells. Despite major progress in the last few years in the understanding of the biology and pathophysiology of the disease, as well as the development of better treatment modalities, CLL remains incurable in most patients, and even control of the disease requires aggressive treatment with significant side effects. A better understanding of the cellular events involved in the pathogenesis and progression of the disease should lead to more targeted and less toxic therapies, with early treatment in patients at risk, possibly enabling cure.
Previous studies have shown that CLL lymphocytes express relatively large amounts of the CD74 (invariant chain; Ii) mRNA compared to normal B cells, and this receptor regulates cell survival in an IL-8 dependent manner.
CD74 is a non-polymorphic type II integral membrane protein that is expressed on antigen presenting cells, including macrophages and B cells. It has a short N-terminal cytoplasmic tail of 28 amino acids (aa), followed by a single 24 aa transmembrane region, and a lumenal domain of approximately 150 aa. The CD74 chain was initially thought to function mainly as an MHC class II chaperone, which promotes ER exit of MHC class II molecules, directs them to endocytic compartments, prevents peptide binding within the ER, and contributes to peptide editing in the MHC class II compartment. A small proportion of CD74 is modified by the addition of chondroitin sulfate (CD74-CS), and this form of CD74 is expressed on the surface of antigen presenting cells. It was previously shown that macrophage migration inhibitory factor (MIF) binds to the CD74 extracellular domain on macrophages, a process that results in the initiation of a signaling pathway in these cells. MIF promotes monocyte/macrophage activation and is required for the optimal expression of TNF, IL-1 and PGE2. MIF-activated macrophages are more phagocytic and better able to destroy intracellular pathogens, such as Leishmania. 
CD44 is a broadly-expressed single-pass transmembrane protein with known kinase-activating properties.
Recently, CD44 was described as an integral component of the CD74 receptor complex. While CD74 is sufficient for the binding of MIF to the cell surface, CD44 was found to be necessary for MIF signal transduction. CD74 expressed on B cells is directly involved in shaping the B cell repertoire by regulating mature B cell survival [Shachar et al. Science. 1996;274:106-108; Matza et al. Proc Natl Acad Sci USA. 2002; 99:3018-3023; Matza et al. 2003 Trends Immunol. 24:246-248] through a pathway leading to the activation of transcription mediated by the NF-κB p65/RelA homodimer and its co-activator, TAFII105 [Matza et al. J Biol Chem. 2001; 276:27203-27206]. NF-κB activation is mediated by the cytosolic region of CD74 (CD74-ICD), which is liberated from the membrane [Matza et al. Immunity. 2002; 17:549-560]. Following the removal of the CD74 lumenal domain, an intramembranal cleavage event occurs at amino acid 42, resulting in the release of the CD74 cytosolic fragment (CD74-ICD; aa 1-42). CD74-ICD then translocates to the cell nucleus and activates NF-κB [Becker-Herman Cell. 2005; 16:5061-5069]. Thus, following this processing step, CD74 acts as a signaling molecule that induces accumulation of mature B cells. This signal is attenuated by degradation of the active CD74-ICD fragment, and its removal from the cytoplasm. Moreover, CD74 stimulation by MIF leads to NF-κB activation, enabling entry of the stimulated B cells into the S phase, an increase in DNA synthesis, cell division, and augmented expression of anti-apoptotic proteins in a CD44 dependent manner. These findings indicate that surface CD74 functions as a survival receptor.
Interestingly, both MIF and CD74 have been associated with tumor progression. It was reported that MIF mRNA is over-expressed in various tumors and MIF has also been associated with the growth of malignant cells [Bando et al. Jpn J Cancer Res. 2002; 93:389-396; Nishihira et al. Ann NY Acad Sci. 2003; 995:171-182]. Many studies have demonstrated the overexpression of CD74 in various cancers including CLL [Narni et al. Blood. 1986; 68:372-377]. CD74 expression in many of these cancers has been suggested to serve as a prognostic factor, with higher relative expression of CD74 behaving as a marker of tumor progression [Mizue et al. Int J Mol Med. 2000; 5:397-403]. Activation of CD74 by MIF on B-CLL cells, initiates a signaling cascade that contributes to tumor progression. This pathway induces NF-κB activation, resulting in the secretion of interleukin 8 (IL-8), which in turn promotes cell survival. Blocking of this pathway leads to decreased cell survival. Thus, CD74 expressed on the surface of B-CLL cells plays a critical role in regulating the survival of these malignant cells [Binsky et al. Proc Natl Acad Sci USA. 2007; 104:13408-13413]. Molecules which participate in CD74 signaling may thus be used as new targets and generation of therapeutic platforms for the treatment of chronic lymphoid leukemia.
CD84 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface molecules. It is a single chain cell-surface protein with an extracellular portion of 199 aa, which contains four potential N-glycosylation sites. The transmembrane region consists of 25 aa, and the 83 aa cytoplasmic tail contains four tyrosines [delaFuente et al. Blood. 1997; 90:2398-2405]. The human CD84 is 57.3% identical to murine CD84. CD84 is predominantly expressed by B cells, T cells, platelets, monocytes, dendritic cells (DCs), and CD84 is also expressed early in hematopoiesis [Calpe et al. Advances in Immunology, Vol 97. 2008; 97:177-250].
Based on the expression of CD84, B cells can be subdivided into CD84hi and CD84lo populations. The CD84hi population represents a subset of memory B cells, which are characterized by co-expression of CD27, somatically mutated Ig variable region genes, and vigorous proliferation in response to CD40L and IL-4, compared to CD84lo B cells.
Nevertheless, the role of CD84 in the biology of these cells is not known. A striking feature of human CD84 is the expression of a complex series of isoforms with several cytoplasmic tails in tumor cells [Palou et al. Tissue Antigens. 2000; 55:118-127]. The expression and functional relevance of these variants are still unknown. Two CD84 transcripts have been described to date in murine tissues. CD84 strongly self-associates with a Kd in the submicromolar range; the association is driven by the Ig-V domain, forming an orthogonal homophilic dimer.